Solid elements serve a variety of functions in electrophoretic and chromatographic systems. In some systems, solid elements serve as sites for the partitioning of solutes, whereas in others they serve as retaining walls for housing separation media. Solid elements thus occur as particles, tubes and plates, depending on the separation mechanism to be employed as well as the arrangement, size and shape of the separation medium. Examples of separation systems using solid elements are affinity chromatography, reversed-phase chromatography, ion exchange chromatography, size exclusion chromatography, and the various forms of electrophoresis, including slab gel electrophoresis, tube gel electrophoresis, capillary electrophoresis (both gel and solution types), isotachophoresis and isoelectric focusing. In some cases, the solid element plays an active role in the partitioning, and in other cases, a passive role.
A phenomenon which occurs in many of these systems, particularly those in which the solid element is a silica-containing material, is electroendosmosis, also referred to as electroosmotic flow, which arises from an electrokinetic potential existing between the wall of the solid element and the liquid or gel separation medium adjacent to the wall. The flow which is caused by this potential is a bulk flow which occurs when an electric field tangential to the solid surface is imposed on the separation medium. In many systems, this bulk flow is considered an interference with the separation process.
While electroosmotic flow can occur in any of these configurations, it is particularly troublesome in capillaries due to their high ratio of wall surface area to internal volume, and to the close proximity of the wall to the sample components being separated. Capillaries are particularly significant since they permit the analysis of extremely small samples with on-line spectroscopic detection, as well as the use of high voltages, thereby achieving separations at high speed.
Accordingly, the suppression of electroosmotic flow in chromatographic, and particularly electrophoretic, systems is one of the goals addressed by the present invention.
Also addressed by this invention is a phenomenon encountered in the separation of proteins by such techniques. Proteins have an inherent tendency to adsorb to silica surfaces. In most separation processes, this adsorption is undesirable, since it leads to peak broadening and asymmetry, and is thereby detrimental to resolution, lowering the accuracy and reproducibility of analyses.
Protein adsorption is of particular concern in systems which are susceptible to electroosmotic flow, since the adsorbed protein affects the wall characteristics, including the zeta potential. Changes in the quantity or distribution of adsorbed protein on the wall will cause the electroosmotic contribution of the flow to vary, both within a single run and between successive runs, further aggravating the difficulties in performing reliable and meaningful comparisons and determinations. Again, these concerns are particularly acute in capillary systems, due to the capillary geometry and the high influence of the capillary wall.
Various methods of reducing or eliminating protein adsorption by silica surfaces are reported in the literature. In general, these methods involve one of two approaches:
(1) creating a Coulombic repulsion between the proteins and silica by appropriate selection of buffer pH and ionic strength; and PA1 (2) chemically bonding a neutral material to the silica surface to eliminate the surface charges which function as adsorption sites.
Examples of the first approach are reported by McCormick, R. M., Anal. Chem. 60:2322-2328 (1988), who describes the use of low pH phosphate buffers to reduce the negative charge of fused silica and distribute phosphate groups over the silica surface as a form of protective screening. The use of high pH buffers with added ionic modifiers is reported by Lauer, H. H., et al., Anal. Chem. 58:166-170 (1986), and Walbroehl, Y., et al., J. Microcolumn Sep. 1:41-45 (1989). As reported by these authors, the buffers convert the proteins to negatively charged species which are repelled by the negatively charged capillary walls.
The second approach was adopted by Jorgenson, J. W., et al., Science 222:266-272 (1983), who bonded glycol-containing materials to fused silica. Hjerten, S. J., J. Chromatogr. 347:191-198 (1985) reported the use of methylcellulose and non-crosslinked polyacrylamide bonded through an organosilane reagent. The use of a poly(vinylpyrrolidinone) coating, applied by way of organosilane surface derivatization was reported by McCormick, referenced above, and the use of a polyethylene glycol coating is reported by Bruin, G. J. M. et al., J. Chromatogr. 471:429-436 (1989).
While each of these approaches has certain merits, they suffer disadvantages as well, particularly due to limits on their ranges of applicability. Approaches involving manipulation of buffer pH and ionic strength are limited in terms of the range of pH under which the separation can be performed, and hence the proteins which can be separated. Approaches involving coating of the silica surface encounter problems in long-term stability, particularly under alkaline conditions. The widely used technique of bonding through siloxane (Si--O--Si--C) bonds, for example, is prone to nucleophilic cleavage under basic conditions.
For protein separation, it is important that one be able to select from a wide range of buffers and pH values because of the vast differences among proteins and the strong influence of pH on the charges of protein molecules, and hence on their migration characteristics. Certain mixtures are best separated at low pH (below the isoelectric point of the proteins), while others afford better separations at pH values above the protein isoelectric points. The ideal system will therefore be one which is both stable and capable of use in both high and low pH regimes.